Many properties of a cell including its shape, internal organization, motility and adhesion depend on a complex network of cytoplasmic protein filaments called the cytoskeleton. The types of filaments comprising the cytoskeleton are actin filaments (microfilaments), microtubules (MTs) and intermediate filaments (IFs). In addition to the three types of filaments, the cytoskeleton contains a number of accessory proteins including vinculin, talin and .alpha.-actinin which link different filaments and connect the cytoskeleton to the plasma membrane (Alberts et al., (1983) Molecular Biology of the Cell, 549-609). The contents of this chapter are hereby incorporated by reference.
Actin filaments and microtubules are dynamic structures, able to rapidly assemble and disassemble to promote processes including muscle contraction and mitosis. IFs, intermediate in diameter (8-10 nm) between actin filaments and microtubules, are concentrated in areas of a cell subject to mechanical stress, such as between adjacent sarcomeres in muscle cells, and are the most stable and least soluble type of filament in the cell.
There are five distinct types of IFs which are expressed in different cell types: type I and II keratins in epithelial cells; type III IFs consisting of vimentin, desmin, glial fibrillary acidic protein and peripherin; type IV neuronal IFs (the neurofilament triplet protein) and type V nuclear lamins (Steinert and Roop (1988) Annu. Rev. Biochem., 57, 593-625).
Long believed to be static, uninteresting structures, IFs are now emerging as dynamic entities. The phosphorylation-mediated reversible disassembly of both type V nuclear lamins and type III vimentin IFs during mitosis has been demonstrated (Dessev et al., (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 2994-2998). In addition, the dynamic phosphorylation of the human IF keratin 1 (type II) chain has also been described (Steinert, (1988) J. Biol. Chem. 263, 13333-13339). When epithelial cells were transfected with a mutant keratin gene, the protein was redistributed throughout the cytoskeleton after 4 days (Albers and Fuchs, (1989) J. Cell Biol., 108: 1477-1493). Biotinylated keratin, when microinjected into primary mouse epidermal (PME) or kangaroo rat kidney epithelial (PtK.sub.2) cells in vivo, incorporated rapidly (between 15 and 30 min) into keratin filament bundles called tonofilaments (Miller et al., (1991) J. Cell Biol., 113: 843-855). The above data suggest that keratin IFs are dynamic structures in vivo.
All keratin chains, as well as other IF types, have a central, highly-conserved .alpha.-helical "rod" domain in regard to size, organization and likely secondary structure flanked by amino- and carboxy-"end" domains (E1, V1, H1 and E2, V2, H2). The V sequences are of highly variable size and amino acid sequence. The H1 and H2 sequences which flank the beginning and end of the rod domain, respectively, (FIG. 1) are highly conserved in sequence. In addition, there are highly conserved canonical-sequences (1A and 2B) at the beginning and end of the rod domain which are common to all IF types; however, the H1 and H2 sequences are unique to keratin IFs (Steinert and Roop, (1988) Annu. Rev. Biochem., 57: 593-625).
Keratin intermediate filaments contain type I (acidic) and type II (neutral-basic) IF proteins. Both type I and type II chains have been characterized by isoelectric point, sequence similarities and immunoreactivity and are required for 10 nm filament formation at the heterodimer level. IFs can comprise up to 95% of total cell protein in keratinocytes.
Tumor necrosis factor (TNF) has been shown to alter the cytoskeletal organization of kidney mesangial cells (Camassi et al. (1990), Kidney Intl., 38: 795-802). Within one to two hours after TNF administration the cells retracted and lost reciprocal contacts. Similar changes were observed after a five minute treatment with platelet activating factor (PAF).
Chipev et al. (Cell, 70: 821-828, (1992)) steadied epidermolytic hyperkeratosis, an autosomal dominant disease affecting the suprabasal level of the epidermis. They found that synthetic peptides corresponding to a region of the conserved H1 domain drastically affected the structural integrity of, or even totally disassembled, preformed filaments in vitro.
Hatzfeld and Weber (J. Cell Biol., 116: 157-166, (1992)) showed that a synthetic peptide corresponding to a portion of the carboxy-terminal end of the "rod" domain (2B region) inhibited IF assembly and disassembled preformed filaments in vitro, while Kouklis et al. (J. Cell Sci., 102: 31-41, (1992)) demonstrated that a 20-residue synthetic peptide containing the same consensus sequence (LLEGE) was able to inhibit assembly of vimentin filaments. In addition, they showed that a monoclonal antibody directed against the 2B sequence, when microinjected into interphasic 3T3 cells, resulted in disruption of vimentin IFs. Stappenbeck and Green (J. Cell Biol., 116: 1197-1209, (1992)) showed that peptides derived from desmoplakins I and II, components of the desmosome which act as a cell attachment site for IFs, promoted the disruption of IFs in COS-7 and 3T3 cells. Finally, Letai et al. (J. Cell Biol., 116: 1181-1195) demonstrated that proline mutations at the ends of the rod domain of keratin IFs destabilized these filaments.
Although the above findings indicated that a peptide corresponding to a sequence at the end of the keratin chain "rod" domain promoted rapid filament disassembly in vitro, the peptides used in vitro and in vivo in the present invention correspond to different regions of the keratin chain. In addition, the use of the H1 and 1A keratin 1 chain peptides for in vivo disruption of keratin and vimentin IFs has not been previously demonstrated.
Steinert et al. (J. Invest. Dermatol., 100: 500, (1993)) reported that synthetic peptides corresponding to certain portions of the keratin 1 chain have significant effects on the structural organization and integrity of filaments both in vivo and in vitro. More specifically, peptides corresponding to the H1, 1A, and 2B regions of the human keratin 1 chain (FIG. 1) resulted in a rapid, reversible disassembly of the IFs in vitro. Similarly, microinjection of the 1A peptide also caused drastic, reversible disassembly of the endogenous IF network of epithelial cells and fibroblasts, due to disruption of keratin and vimentin IFs, respectively. These sequences are normally located adjacent to each other in the IF and represent key points of interaction in maintenance of IF structure and integrity. IFs are excellent cell type specific markers and represent potential cell type specific targets for drug therapy.